1. ¹Department of Microbiology and Immunology, Medical University of South Carolina, Charleston, SC 29425, USA
2. ²Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC 29425, USA
3. ³Department of Urology, Medical University of South Carolina, Charleston, SC 29425, USA
4. ⁴Cancer Gene Therapy Group, GKT Dental Institute, King's College London, London SE5 9NU, UK

Correspondence: James S. Norris, Department of Microbiology and Immunology, P.O. Box 250504, 173 Ashley Avenue, Charleston, SC 29425 USA. Fax: +1 843 792 4882. E-mail: norrisjs@musc.edu

Received 12 September 2005; Revised 20 June 2006; Accepted 4 July 2006.

Abstract

The potential anti-tumor agent Apoptin activates apoptosis in many human cancers and transformed cell lines, but is believed to be less potent in primary cells. Although caspase 3 is activated during apoptin-induced apoptosis, the mechanism of tumor cell killing remains elusive. We now show that apoptin-mediated cell death involves modulation of the sphingomyelin–ceramide pathway. Treating cells with Ad-GFPApoptin resulted in increased ceramide accumulation and enhanced expression of acid sphingomyelinase (ASMase) with a concomitant increase in ASMase activity and decreased sphingomyelin. Using confocal microscopy, ASMase, normally present in the endosomal/lysosomal compartment, was observed to translocate to the cell's periphery. Cotreatment of Ad-GFPApoptin-infected cells with the ASMase inhibitor desipramine (2.5 M) attenuated (30%; P < 0.01) apoptin-induced cell death. Apoptin was also able to induce a significant decline in sphingosine content by inhibition of ceramide deacylation through down-regulation of acid ceramidase at the protein level. Supporting the role of ceramide in apoptin action, treatment of cells with the combination of an exogenous cell-permeable ceramide analog (C6-ceramide) and Ad-GFPApoptin infection yielded a significant increase (P < 0.01) in apoptosis over either treatment modality alone. Together, these data suggest that apoptin modulates ceramide/sphingolipid metabolism as part of its mechanism of action.